Tibetan medicine Liuwei Muxiang pills (LWMX pills) effectively protects mice from chronic non-atrophic gastritis.

BACKGROUND: Chronic non-atrophic gastritis (CNG) is the most common type of chronic gastritis. If not actively treated, it may induce gastric cancer (GC). Western medicine is effective in CNG, but there are more adverse reactions after long-term medication, and it is easy to relapse after treatment, which affects patients' health and life. Tibetan medicine Liuwei Muxiang Pills (LWMX pills) is a traditional Tibetan medicine compound, which has a unique curative effect in the treatment of gastric inflammation, especially chronic non-atrophic gastritis. However, the mechanisms of LWMX pills for treatment CNG still remain poor known. PURPOSE: The aim of this study was to evaluate the therapeutic intervention potential of Tibetan medicine LWMX pills on CNG and explore its potential mechanisms in mice models. METHODS: The mice models was established to evaluate the therapeutic effect of LWMX pills on CNG. The main components of LWMX pills were analyzed by GC-MS. HE staining, immunohistochemistry, proteomics and Western Blot were used to analyze the potential mechanism of LWMX pills for CNG treatment. RESULTS: In the present study, LWMX pills containing costunolide, dehydrocostuslactone and antioxidants were found. IF results showed that the expression of ALDH1B1 in the control group was significantly lower than that in the model group in the gastric mucosa tissue, and the expression of ALDH1B1 was significantly lower in the 25 mg/ml LWMX Pills group (one month) and 25 mg/ml LWMX Pills group (two months) than in the model group. IHC revealed that model group samples expressed higher levels of Furin than 25 mg/ml LWMX Pills group samples, as evidenced by very strong staining of Furin in gastric mucosal cells. However, AMY2 staining in gastric mucosal cells did not differ significantly between the treated and control groups. the protein expression levels of these proteins were decreased in 25 mg/mL LWMX pills. Meanwhile, we found that the CAM1 protein expression in the in 25 mg/ml LWMX pills group (two mouths) was increased compared to the in 25 mg/ml LWMX pills group (one mouths).Western blotting showed that the protein expression levels of Furin, AMY2A, CPA3, ALDH1B1, Cam1, COXII, IL-6, IL-1ß were decreased in 25 mg/mL LWMX pills. Meanwhile, that the CAM1 protein expression in the in 25 mg/ml LWMX pills group (two mouths) was increased compared to the in 25 mg/ml LWMX pills group (one mouths). CONCLUSION: 25mg/ml LWMX pill treatment for one month had better therapeutic effect on mice CNG. Further proteomic results showed that LWMX pills maintain gastric function by inhibiting inflammation and oxidative stress, and we also found that LWMX pills regulate the expression of proteins associated with cancer development (Amy2, Furin).

Effects of the Tibetan medicine Byur dMar Nyer lNga Ril Bu on Alzheimer's disease in mice models.

ETHNOPHARMACOLOGICAL RELEVANCE: Byur dMar Nyer lNga Ril Bu (BdNlRB) is a classic Tibetan medicine prescription for treating " white vein disease". Alzheimer's disease (AD) is a chronic degenerative disease of the central nervous system, characterized by distinct "white vein disease". In the absence of effective drugs for AD, BdNlRB may be a possible treatment for AD. AIM OF THE STUDY: To verify the therapeutic effect and possible mechanism of the proved Tibetan medicine BdNlRB on Alzheimer's disease. MATERIALS AND METHODS: 60 APP/PS1 double transgenic AD mice (Mt) and 60 Aß1-40 protein-induced AD mice (Mi) were divided into 3 groups according to the dose of BdNlRB: BdNlRB-100, BdNlRB-200 and BdNlRB-400, with 100, 200 and 400 mg/kg*weight, respectively. The mice were administrated by gavage for 8 weeks. The cognitive ability of mice was detected by Morris Water Maze, the expression of Aß protein, p-tau and microglia was detected by immunofluorescent staining, the protein expression in the hippocampus was detected by proteomics, and the abundance of fecal intestinal flora was detected by 16S RNA. RESULTS: The learning ability and memory ability of Mi mice were significantly improved after BdNlRB administration. The learning ability of Mt mice was significantly improved, while the memory ability was not improved after BdNlRB administration. After the treatment with low and medium doses of BdNlRB, the expression of p-tau decreased significantly (the rate of decrease in BdNlRB-100 and BdNlRB-200 groups was 8.05% and 12.7%, respectively), and the number of microglia increased (39.3% and 31.6%, respectively). BdNlRB significantly affected the protein expression in the hippocampus of Mt mice. 382 proteins in different expression in all three groups mainly involved in amino acid synthesis, fatty acid degradation, glutamine metabolism, synaptic vesicular cycle and oxidative phosphorylation, PPAR signaling pathway and Fc gamma-mediated phagocytosis were activated. Meanwhile, the administration of BdNlRB can regulate the intestinal flora of Mt mice, which reduces the abundance of Muribaculum and uncultured bacteroidales bacterium, and improves the abundance of Ruminococcus-1 and Ruminiclostridium-9. CONCLUSION: The oral administration of BdNlRB significantly improved the cognitive ability of AD mice, and neuroinflammation and intestinal flora regulation were the possible mechanisms.

Targeted Separation of COX-2 Inhibitor from Pterocephalus hookeri Using Preparative High-Performance Liquid Chromatography Directed by the Affinity Solid-Phase Extraction HPLC System.

Pterocephalus hookeri, as a kind of popular traditional Tibetan medicine, is reputed to treat inflammatory related diseases. In the present work, a cyclooxygenase-2 functionalized affinity solid-phase extraction HPLC system was developed and combined with preparative-HPLC for rapidly screening and separating cyclooxygenase-2 ligand from P. hookeri extracts. Firstly, ligands of cyclooxygenase-2 were screened from extracts by affinity solid-phase extraction HPLC system. Then directed by the screening results, the recognized potential active compounds were targeted separated. As a result, the major cyclooxygenase-2 inhibitor of P. hookeri was obtained with a purity of >95%, which was identified as sylvestroside I. To test the accuracy of this method, the anti-inflammatory activity of sylvestroside I was inspected in lipopolysaccharide-induced RAW 264.7 cells. The results show that sylvestroside I significantly suppressed the release of prostaglandin E2 with dose-dependent, which was in good agreement with the screening result of the affinity solid-phase method. This method of integration of screening and targeted separation proved to be very efficient for the recognition and isolation of cyclooxygenase-2 inhibitors from natural products.

Amelioration of diabetic retinopathy in db/db mice by treatment with different proportional three active ingredients from Tibetan medicine Berberis dictyophylla F.

ETHNOPHARMACOLOGICAL RELEVANCE: Berberis dictyophylla F., a famous Tibetan medicine, has been used to prevent and treat diabetic retinopathy (DR) for thousands of years in clinic. However, its underlying mechanisms remain unclear. AIM OF THE STUDY: The present study was designed to probe the synergistic protection and involved mechanisms of berberine, magnoflorine and berbamine from Berberis dictyophylla F. on the spontaneous retinal damage of db/db mice. MATERIALS AND METHODS: The 14-week spontaneous model of DR in db/db mice were randomly divided into eight groups: model group, calcium dobesilate (CaDob, 0.23 g/kg) group and groups 1-6 (different proportional three active ingredients from Berberis dictyophylla F.). All mice were intragastrically administrated for a continuous 12 weeks. Body weight and fasting blood glucose (FBG) were recorded and measured. Hematoxylin-eosin and periodic acid-Schiff (PAS) stainings were employed to evaluate the pathological changes and abnormal angiogenesis of the retina. ELISA was performed to assess the levels of IL-6, HIF-1α and VEGF in the serum. Immunofluorescent staining was applied to detect the protein levels of CD31, VEGF, p-p38, p-JNK, p-ERK and NF-κB in retina. In addition, mRNA expression levels of VEGF, Bax and Bcl-2 in the retina were monitored by qRT-PCR analysis. RESULTS: Treatment with different proportional three active ingredients exerted no significant effect on the weight, but decreased the FBG, increased the number of retinal ganglionic cells and restored internal limiting membrane. The results of PAS staining demonstrated that the drug treatment decreased the ratio of endothelial cells to pericytes while thinned the basal membrane of retinal vessels. Moreover, these different proportional active ingredients can markedly downregulate the protein levels of retinal CD31 and VEGF, and serum HIF-1α and VEGF. The gene expression of retinal VEGF was also suppressed. The levels of retinal p-p38, p-JNK and p-ERK proteins were decreased by drug treatment. Finally, drug treatment reversed the proinflammatory factors of retinal NF-κB and serum IL-6, and proapoptotic Bax gene expression, while increased antiapoptotic Bcl-2 gene expression. CONCLUSIONS: These results indicated that DR in db/db mice can be ameliorated by treatment with different proportional three active ingredients from Berberis dictyophylla F. The potential vascular protection mechanisms may be involved in inhibiting the phosphorylation of the MAPK signaling pathway, thus decreasing inflammatory and apoptotic events.

Acute hepatotoxicity and nephrotoxicity risk assessment of the Tibetan medicine 25 flavors of the turquoise pill based on 1H-NMR metabonomics.

ETHNOPHARMACOLOGICAL RELEVANCE: 25 flavors of the turquoise pill, a traditional Tibetan medicine for the treatment of various types of hepatitis, has not been investigated on its safety, especially the component mineral turquoise, which is believed to be essential but worried for its potential toxicity. AIM OF THE STUDY: To explore the potential acute toxicity and function of 25 flavors of the turquoise pill and turquoise, the possible mechanism of the effects of turquoise and 25 flavors of the turquoise pill were systematically studied based on 1H NMR metabolomics. MATERIALS AND METHODS: The rats were administered with turquoise and 25 flavors of the turquoise pill by gavage for 7 days, and samples of serum, liver, and kidney were collected. The potential toxicity and function of turquoise and 25 flavors of the turquoise pill on the liver and kidney of SD rats were evaluated by 1H NMR metabonomics, histopathology, and biochemical indexes. RESULTS: The results demonstrated that 25 flavors of the turquoise pill could scavenge free oxygen radicals, strengthen aerobic respiration and inhibit glycolysis in the liver. It did not cause oxidative stress in the kidney with no obvious damage. By modulation of branched-chain amino acids (BCAAs), 25 flavors of the turquoise pill can improve the utilization of glucose and promote aerobic respiration of the kidney. CONCLUSION: Considering the high dosage and short duration used in this study relative to their typical clinical usage, administration of 25 flavors of the turquoise pill and its component mineral turquoise are safe to livers and kidneys.

Hypnotic-like Aspects of the Tibetan Tradition of Dzogchen Meditation.

Dzogchen meditation has been practiced by Bonpo and Buddhist yogis for at least 1,200 years. Dzogchen utilizes methods of meditation and yogic exercises that are said to help one fully awaken from illusions of self and reality that cause suffering in life. The philosophy and experiential practice of Dzogchen is very similar to hypnosis. Dzogchen techniques utilize hypnotic-like practices of selective attention, visualization, and posthypnotic suggestion to help yogis experience advanced insights into the nature of mind. The experience of Dzogchen can be likened to the experience of hypnosis in terms of their phenomenological and psychophysiological effects. Finally, there are also many theoretical similarities between aspects of the ego state therapy, neo-dissociation, sociocognitive, and Ericksonian theories of hypnosis with the tradition of Dzogchen meditation.

Inventing a herbal tradition: The complex roots of the current popularity of Epilobium angustifolium in Eastern Europe.

ETHNOPHARMACOLOGICAL RELEVANCE: Currently various scientific and popular sources provide a wide spectrum of ethnopharmacological information on many plants, yet the sources of that information, as well as the information itself, are often not clear, potentially resulting in the erroneous use of plants among lay people or even in official medicine. Our field studies in seven countries on the Eastern edge of Europe have revealed an unusual increase in the medicinal use of Epilobium angustifolium L., especially in Estonia, where the majority of uses were specifically related to "men's problems". THE AIM OF THE CURRENT WORK IS: to understand the recent and sudden increase in the interest in the use of E. angustifolium in Estonia; to evaluate the extent of documented traditional use of E. angustifolium among sources of knowledge considered traditional; to track different sources describing (or attributed as describing) the benefits of E. angustifolium; and to detect direct and indirect influences of the written sources on the currently documented local uses of E. angustifolium on the Eastern edge of Europe. MATERIALS AND METHODS: In this study we used a variety of methods: semi-structured interviews with 599 people in 7 countries, historical data analysis and historical ethnopharmacological source analysis. We researched historical and archival sources, and academic and popular literature published on the medicinal use of E. angustifolium in the regions of our field sites as well as internationally, paying close attention to the literature that might have directly or indirectly contributed to the popularity of E. angustifolium at different times in history. RESULTS: Our results show that the sudden and recent popularity in the medical use of E. angustifolium in Estonia has been caused by local popular authors with academic medical backgrounds, relying simultaneously on "western" and Russian sources. While Russian sources have propagated (partially unpublished) results from the 1930s, "western" sources are scientific insights derived from the popularization of other Epilobium species by Austrian herbalist Maria Treben. The information Treben disseminated could have been originated from a previous peak in popularity of E. angustifolium in USA in the second half of the 19th century, caused in turn by misinterpretation of ancient herbals. The traditional uses of E. angustifolium were related to wounds and skin diseases, fever, pain (headache, sore throat, childbirth), and abdominal-related problems (constipation, stomach ache) and intestinal bleeding. Few more uses were based on the similarity principle. The main theme, however, is the fragmentation of use and its lack of consistency apart from wounds and skin diseases. CONCLUSIONS: Historical ethnobotanical investigations could help to avoid creating repeating waves of popularity of plants that have already been tried for certain diseases and later abandoned as not fully effective. There is, of course, a chance that E. angustifolium could also finally be proven to be clinically safe and cost-effective for treating benign prostatic hyperplasia, but this has not yet happened despite recent intensive research. Documented traditional use would suggest investigating the dermatological, intestinal anti-hemorrhagic and pain inhibiting properties of this plant, if any.

Tsantan Sumtang attenuated chronic hypoxia-induced right ventricular structure remodeling and fibrosis by equilibrating local ACE-AngII-AT1R/ACE2-Ang1-7-Mas axis in rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Tsantan Sumtang, which consists of Choerospondias axillaris (Roxb.) Burtt et Hill, Myristica fragrans Houtt and Santalum album L, is a traditional and common prescription of Tibetan medicine. Tsantan Sumtang originates from Four Tantra with properties of nourishing heart and has been used as a folk medicine for cardiovascular diseases and heart failure in Qinghai, Tibet and Inner Mongolia. Our previous studies found that Tsantan Sumtang showed beneficial effects on right ventricular structure in hypoxia rats, while the underling mechanism remains unclear. AIM OF THE STUDY: To elucidate the underlying mechanisms of Tsantan Sumtang attenuated right ventricular (RV) remodeling and fibrosis of chronic hypoxia-induced pulmonary arterial hypertension (HPAH) rats. MATERIALS AND METHODS: Fifty male Sprague Dawley (SD) rats (170 ± 20 g) were randomly divided into control group, hypoxia group, and hypoxia + Tsantan Sumtang groups (1.0 g·â€¯kg-1·day-1, 1.25 g·â€¯kg-1·day-1, 1.5 g ·kg-1·day-1). Rats in the hypoxia group and hypoxia + Tsantan Sumtang groups were maintained in a hypobaric chamber by adjusting the inner pressure and oxygen content to simulate an altitude of 4500 m for 28 days. The mean pulmonary arterial pressure (mPAP), right ventricle hypertrophy index (RVHI), the ratio of RV weight to tibia length (TL) (RV/TL), heart rate (HR) and RV systolic pressure (RVSP) was determined. Histomorphological assay of RV structure was evaluated by hematoxylin and eosin (HE) staining. RV tissue fibrosis was assessed by collagen proportion area (CPA), collagen I, collagen III and hydroxyproline content. CPA was obtained by picro-sirius red staining (PSR). The expression of collagen I and collagen III were detected by immunohistochemistry and western blotting. The hydroxyproline content was detected by alkaline hydrolysis. In addition, the level of angiotensin II (AngII) and angiotensin 1-7 (Ang1-7) in RV tissue was tested by enzyme-linked immune sorbent assay (ELISA). Protein expression of angiotensin-converting enzyme (ACE), AngII, AngII type 1 receptor (AT1R), angiotensin-converting enzyme 2 (ACE2), Mas receptor (Mas) were determined by immunohistochemistry and western blotting. mRNA level of ACE, AT1R, ACE2, Mas were tested by qPCR. The chemical profile of Tsantan Sumtang was revealed by UHPLC-Q-Exactive hybrid quadrupole-orbitrap mass analysis. RESULTS: Our results showed that RVHI, RV/TL and RVSP were significantly increased in HPAH rat. Furthermore, levels of collagen I, collagen III and hydroxyproline were up-regulated in RV tissue under hypoxia. We found that RV hypertrophy and fibrosis were associated with increased expression of ACE, AngII, AT1R as well as decreased expression of ACE2, Ang1-7 and Mas. RV remodeling and fibrosis were attenuated after Tsantan Sumtang administration by up-regulating ACE2 and Mas level as well as down-regulating ACE, AngII and AT1R levels in RV tissue. 35 constituents in Tsantan Sumtang were identified. CONCLUSION: Tsantan Sumtang attenuated RV remodeling and fibrosis in rat exposed to chronic hypoxia. The pharmacological effect of Tsantan Sumtang was based on equilibrating ACE-AngII-AT1R and ACE2-Ang1-7-Mas axis of RV tissue in HPAH rat.

Longzhibu disease and its therapeutic effects by traditional Tibetan medicine: Ershi-wei Chenxiang pills.

ETHNOPHARMACOLOGICAL RELEVANCE: Ershi-wei Chenxiang pills (ECP) or Aga Nixiu wan (ཨ་གར་ཉི་ཤུ།), composed of 20 Tibetan medicines, has the effect of promoting blood circulation to remove blood stasis. As a common and frequent prescription used by traditional Tibetan medicine in clinical treatment of Longzhibu disease (cerebral ischemia sequelae), it has a significant effect. However, its anti-cerebral ischemia mechanism is still unclear. MATERIALS AND METHODS: The chemical components of ECP were determined by high-performance liquid chromatography and gas chromatography-mass spectrometry. SD rats were randomly divided into Sham, MCAO, Nim (20.00 mg/kg), and ECP (1.33 and 2.00 g/kg) groups, with 13 animals in each group. After 14 days of oral administration, we established a model of cerebral ischemia reperfusion injury by blocking the middle cerebral artery of rats. After 24 h of reperfusion injury, we evaluated the protective effect of ECP on ischemic brain by neural function score, TTC, H&E and Nissl staining. TUNEL fluorescence, western blot and immunohistochemistry were used to detect the phenomenon of apoptosis and the expression of apoptosis-related proteins Bax, Bcl-2, Cyto-c and activated Caspase-3. Furthermore, western blot, qRT-PCR and immunohistochemistry were employed to detect CaMKⅡ, ATF4 and c-Jun gene and protein expression. RESULTS: ECP contains agarotetrol, eugenol, oleanolic acid, ursolic acid, dehydrodiisoeugenol, hydroxysafflor yellow A, kaempferide, gallic acid, alantolactone, isoalantolactone, costunolide, dehydrocostus lactone, brucine, strychnine, echinacoside, bilirubin and cholic acid. Compared with MCAO group, ECP can significantly ameliorate the neurological deficit of cerebral ischemia in rats and reduce the volume of cerebral infarction. Pathological and Nissl staining results showed that ECP sharply inhibited the inflammatory infiltration injury of neurons and increased the activity of neurons in comparation with the MCAO group. TUNEL fluorescence apoptosis results confirmed that ECP obviously inhibited the apoptosis of neurons. Meanwhile, the results of immunohistochemistry and western blot demonstrated that EPC can dramatically inhibit the expression of pro-apoptotic proteins Bax, Cyto-c and activated Caspase-3, while increase the level of anti-apoptotic protein Bcl-2. In addition, compared with MCAO group, CaMK Ⅱ gene and protein expression were improved significantly by ECP administration. while, the expression of ATF4 and c-Jun genes and proteins were decreased. CONCLUSIONS: In conclusion, this study preliminarily demonstrated that the protective effect of ECP on ischemic brain is related to the improvement of neurological deficit, reducing the size of cerebral infarction, improving the activity of neurons, inhibiting the mitochondrial apoptosis pathway by regulating the protein expression of CaMKⅡ, ATF4 and c-Jun. However, further in vivo and in vitro investigations are still needed to clarify the underlying mechanism of ECP in treating cerebral ischemia sequelae.

Medicinal and edible plants used by the Lhoba people in Medog County, Tibet, China.

ETHNOPHARMACOLOGICAL RELEVANCE: The Lhoba people are a small, ancient, tribal ethnic group from the Himalayas and are located in the Tibet Autonomous Region of China. Medog County is rich in biocultural diversity. For a long time, Medog has been almost isolated from the outside world. The Lhoba people, who live in Medog, have maintained a relatively unique lifestyle and have accumulated rich traditional knowledge (TK), especially about medicinal and edible plants. Currently, there is very little documentation of the plants traditionally used by the local Lhoba communities. AIM OF THE STUDY: Our investigation aimed to (i) document the species of medicinal and food plants used by the Lhoba people in Medog County, Tibet, China; (ii) screen the most important plant taxa for specific medicines, and identify the aliments treated to further contribute to drug and food supplement research; and (iii) examine whether the ethnobotanical knowledge of the Lhoba is similar among different tribes and discuss traditional uses in the health practices and livelihoods of the local communities. METHODS: Ethnobotanical data were recorded through semi-structured interviews, guided field trips, and quantitative analysis. The informant consensus factor (FIC) was used as a quantitative index. RESULTS: Ninety-one informants (61 men and 30 women) were interviewed. A total of 75 species, including 37 medicinal plants for 14 categories of diseases and 57 edible plants from six types of food, were recorded. Among the usage types of medicinal plants, the highest FIC values were recorded for antidotes (FIC = 0.98), anthelmintics (FIC = 0.98), and treatments of gastrointestinal problems (FIC = 0.93). The FIC values for different types of edible plants were very similar. The most frequently used medicinal and food plants in the studied communities are Zanthoxylum motuoense, Crassocephalum crepidioides, and Swertia nervosa. According to the comparative study, few differences in the use of wild plants were found. There appeared to be more overlapping species between two Lhoba tribes in Medog, named Mixingba and Miguba, with 46 (61%) common species, compared with the Bo'gaer tribe in Milin, which had only two (2.7%) overlapping species. This might be due to the different geographical environments, vegetation types, and different influences of other ethnic cultures. CONCLUSIONS: The Lhoba people in Medog County, Tibet, China, have rich TK about the uses of wild plants. However, the TK is seriously threatened due to environmental degradation and acculturation, and it showed signs of being forgotten and abandoned by the younger generation. Therefore, measures are urgently needed to document and protect the TK of the uses of the wild plant resources; and (i) the most frequently used medicinal and/or edible plants; (ii) the plants used to treat the most commonly mentioned diseases; and (iii) the endemic species that are widely used in Medog, which should be assessed for their potential future as food supplements and therapeutic products.

Tibetan medicine Ru-yi-Zhen-bao Pills exhibits anti-migraine effect through mediating PAG anti-nociceptive channel.

ETHNOPHARMACOLOGICAL RELEVANCE: Migraine is a disabling neurovascular disorder, which increases risk of cardiovascular events and is a social burden worldwide. The present first-line anti-migraine medications can cause overwhelming side-effects, of which one includes the onset of cardiovascular disease. As one of the marketed Tibetan drugs, Ru-yi-Zhen-bao Pills (RYZBP) have been clinically used to treat cardiovascular disorders and as anti-migraine medication. However, there is currently no research exploring the anti-migraine actions of RYZBP. AIM OF THE STUDY: The current research was designed to assess the anti-migraine roles of RYZBP and explore the underlying mechanisms in a nitroglycerin (NTG)-induced migraine rat model trial. MATERIALS AND METHODS: 120 rats were randomly divided into the following six groups of 20 rats each: normal control group, model control group, positive control group, and RYZBP high/medium/low-dose groups (Ru-yi-Zhen-bao Pills; TH 1.00 g/kg, TM 0.50 g/kg and TL 0.25 g/kg). All rats were administered intragastrically for 7 consecutive days, which were subcutaneously injected with the NTG (10 mg/kg) after the last gavage (except in the normal control group). 3min after NTG treatment, 30 rats (5 rats from each group) were anesthetized and devoted to electroencephalogram(EEG) testing, which was used to evaluate the analgesic effect of RYZBP. One hour after NTG treatment, the rest of the 90 rats (15 rats from each group) were anesthetized and midbrain tissue sample was dissected. The dissection was then washed with physiological saline and collected. The histopathological changes in the periaqueductal gray(PAG) of 5 tissue samples were determined by aematoxylin-eosin (H&E) staining, as well as an estimation of substance P (SP) and neurokinin 1 receptor (NK1R) expression through immunohistochemically staining(IHC). Another 5 midbrain preparations were carried out to evaluate calcitonin gene-related peptide (CGRP), proenkephalin (PENK), SP, and cholecystokinin (CCK) expressions by real-time quantitative polymerase chain reaction (RT-qPCR). The rest of the 5 brainstem tissues were then used to measure CCK, CGRP, and opioid peptide receptor (DORR) levels by western blotting(WB). RESULTS: In the EEG test, RYZBP (TM 0.50 g / kg) treatment transformed the EEG pain-wave of the NTG-induced migraine model rats in different time period. In the mechanism assay, compared with the model control group, RYZBP pretreatment reduced inflammatory cell infiltration, fibrosis and vacuolation of neuronal cells of PAG tissue seen by HE staining. IHC experiments further showed that RYZBPTM up-regulated SP expression levels and enhanced NK1R levels in the NTG-induced migraine rats (P < 0.05). Therapeutic administration of RYZBP also increased PENK mRNA expression and DORR protein level. Both RT-qPCR and western blotting trials indicated that RYZBP treatment significantly decreased CCK and CGRP expression levels (P < 0.01 or P < 0.05) in the NTG-induced migraine rats. CONCLUSIONS: RYZBP has the potential to be an effective anti-migraine treatment through suppressing the EEG pain-wave, increasing the levels of SP, PENK, DORR and reducing expression of CCK and CGRP. Mediating the PAG anti-nociceptive channel and inhibiting central sensitization were the two potential mechanisms, which offers further evidence for clinical therapy.

Traditional Tibetan medicine Anzhijinhua San attenuates ovalbumin-induced diarrhea by regulating the serotonin signaling system in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Tibetan medicine has been practiced for 3800 years. Anzhijinhua San (AZJHS), which is a traditional Tibetan medicine, has been effective in the treatment of indigestion, anorexia and cold diarrhea. However, the effects of AZJHS on allergic diarrhea have not been reported. AIM OF THE STUDY: The aim of the present study was to elucidate the effect of AZJHS on experimental ovalbumin-induced diarrhea and elucidate its possible mechanism. MATERIALS AND METHODS: Female BALB/c mice were sensitized by intraperitoneal injection with 50 µg ovalbumin (OVA) and 1 mg alum in saline twice during a 2-week period. From day 28, mice were orally challenged with OVA (50 mg) every other day for a total of ten times. AZJHS (46.8 and 468.0 mg/kg) was orally administered every other day from day 0-46. Food allergy symptoms were evaluated. OVA- specific IgE, 5-HT and its metabolites in serum were determined. Immunohistochemical and histopathology were performed in gastrointestinal tract tissues. 5-HT-related gene expression was assayed in the colon. RESULTS: Severe symptoms of allergic diarrhea were observed in the model group (diarrhea, anaphylactic response, and rectal temperature). AZJHS (46.8 and 468.0 mg/kg) significantly reduced mouse diarrhea and significantly prevented the increases in OVA-specific IgE levels (P < 0.05), which challenge with OVA. AZJHS (46.8 and 468.0 mg/kg) significantly prevented the increases in 5-HT-positive cells. The nuclei of EC cells in the AZJHS (46.8 and 468.0 mg/kg) group increased in size and the secretory granules were fewer in number compared with those in the model group. AZJHS (46.8 and 468.0 mg/kg) significantly increased the relative fold changes of 5-HTP and 5-HT compared with the model group. The mRNA expression of the serotonin transporter (Sert) and serotonin receptor 3A (Htr3a) was significantly decreased after the 10th challenge with OVA, and AZJHS (46.8 and 468.0 mg/kg) significantly increased these levels. CONCLUSIONS: We demonstrated that the administration of AZJHS attenuated OVA-induced diarrhea by regulating the serotonin pathway. These results indicated that AZJHS may be a potential candidate as an anti-allergic diarrhea agent.

Srolo Bzhtang, a traditional Tibetan medicine formula, inhibits cigarette smoke induced airway inflammation and muc5ac hypersecretion via suppressing IL-13/STAT6 signaling pathway in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Srolo Bzhtang (SBT), a traditional Tibetan medicine formula, was composed of three herbs, Solms-Laubachia eurycarpa, Bergenia purpurascens, Glycyrrhiza uralensis, and one lac, and was first documented in the ancient Tibetan medical work Four Medical Tantras (rGyud-bzhi) in the eighth century AD. It has been widely used to treat lung "phlegm-heat" syndromes such as chronic bronchitis and chronic obstructive pulmonary disease (COPD). OBJECTIVE: The aim of this study was to evaluate the potential influences of aqueous extract of SBT on airway inflammation and mucus secretion and to reveal the underlying mechanism in a rat model of cigarette smoke (CS)-induced chronic bronchitis (CB). MATERIALS AND METHODS: Sixty male Sprague-Dawley rats were randomly divided to six groups: control (room air exposure), model (CS exposure), DEX (CS exposure and 0.2 mg/kg/day dexamethasone), and three SBT (CS exposure and 1.67, 2.50, and 3.34 g/kg/day SBT) groups. DEX and the three doses of SBT were administered by oral gavage every day for eight weeks. Pathological changes and mucus expression in the lung tissue were determined by hematoxylin and eosin (H&E), Alcian blue-periodic acid-Schiff (AB-PAS) and immunohistochemical staining. The levels of cytokines in bronchoalveolar lavage fluid (BALF) were assessed by ELISA. Western blot analysis and qRT-PCR were performed to explore the effects of SBT on the expression of IL-13, STAT6 and MUC5AC. RESULTS: Pretreatment with SBT attenuated the TNF-α, IL-8, IL-13 expression levels in BALF and the inflammatory cell infiltration in bronchial walls and peribronchial lung tissue. SBT exhibited a dose-dependent downregulation of MUC5AC expression as assessed by AB-PAS and immunohistochemical staining. The protein and mRNA levels of IL-13, STAT6/p-STAT6 and MUC5AC were also downregulated by SBT preconditioning. CONCLUSION: These results for the first time demonstrated that SBT exhibited protective effects on CS-induced airway inflammation and MUC5AC hypersecretion, which might be related to the downregulation of the IL-13/STAT6 signaling pathway.

Study on anti-hyperuricemia effects and active ingredients of traditional Tibetan medicine TongFengTangSan (TFTS) by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry.

TongFengTangSan (TFTS), a traditional Tibetan medicine comprising of Tinospora sinensis (TS), Terminalia chebula Retz (TC) and Trogopterori faeces (TF), is used to treat joint diseases like gout, gout arthritis, swelling, pain etc. Despite the significant therapeutic effects of TFTS, its pharmacological components have not been analyzed so far. Therefore, the chemical composition of the effective part of TFTS was analyzed by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). The results show that the ethanol extract (EE) of TFTS was more effective in reducing the serum uric acid (SUA) and XOD (Serum and Liver) levels in a hyperuricemic rats model compared to the TFTS raw powder (RP). UHPLC-Q-TOF-MS/MS identified a total of 106 compounds in the positive and negative ion mode, of which 87 were from TC, 13 from TS and 6 from TF. In addition, 106 compounds contained 57 tannins, 6 triterpenoids, 10 alkaloids, 7 flavonoids, 22 organic acids and 4 phenylpropanoids. The preliminary results indicate that the EE of TFTS includes the active anti hyperuricemic substances. The present study first investigated the efficacy and the active components of TFTS in hyperuricemic treatment, and further summarized the diagnostic ion and neutral loss patterns of MS/MS cracking of tannic compounds. These findings lay the foundation for the further study and clinical application of TFTS.

Evaluation of hepatotoxicity potential of a potent traditional Tibetan medicine Zuotai.

ETHNOPHARMACOLOGICAL RELEVANCE: Zuotai (gTso thal) has a long history in the treatment of cardiovascular disease, liver and bile diseases, spleen and stomach diseases as a precious adjuvant in Tibetan medicine. However, Zuotai is a mercury preparation that contains 54.5% HgS. Its application has always been controversial. AIM OF THE STUDY: To evaluate the toxicological effects of Zuotai in hepatocytes and in zebrafish. MATERIALS AND METHODS: MTT was used to determine the survival rate of hepatocytes; Hoechst and TUNEL staining were used to detect the apoptosis cells; Western blot and RT-qPCR assay were used to determine the expression levels of the protein and mRNA; Liver morphology observation and H&E staining were used to evaluate the hepatotoxicity of Zuotai in Zebfrafish. RESULTS: The survival rate of L-02 cells, HepG2 cells and RBL-2A cells reduced by Zuotai (10-4-0.1 mg/mL) in a dose and time-dependent manner. Zuotai (0.1 mg/mL) induced HepG2 cells shrinkage, condensation and fragmentation and increased the number of apoptosis cells. The protein expression levels of cleaved Caspase-3 and Bax were increased and the expression levels of Bcl-2 were reduced after HepG2 cells exposed to Zuotai (10-4-0.1 mg/mL) for 24 h. In addition, Zuotai (0.2 mg/mL) induced the darker liver color of the larval zebrafish and changed the liver morphologic of adult zebrafish. Zuotai (0.2 mg/mL) also increased the mRNA levels of CYP1A1, CYP1B1 and MT-1 in the liver of adult zebrafish. However, no significantly hepatotoxicity was observed after hepatocytes and zebrafish exposed to HgS at the same dose. CONCLUSIONS: Results showed that Zuotai induced hepatotoxicity effectively under a certain dose but its hepatotoxicity likely occurs via other mechanisms that did not depend on HgS.

Amelioration of diabetic nephropathy in db/db mice treated with tibetan medicine formula Siwei Jianghuang Decoction Powder extract.

Siwei Jianghuang Decoction Powder (SWJH) documented originally in the Four Medical Tantras-Blue Glaze exhibited beneficial effects on diabetic nephropathy (DN) via combined synergistically action of multiple formula components including Curcumae longae Rhizoma, Berberidis dictyophyllae Cortex, Phyllanthi Fructus and Tribuli Fructus. This study investigated the effects of SWJH on DN in db/db mice and possible underlying mechanisms. The ten weeks old db/db mice treated with SWJH by intra-gastric administration once a day for 8 weeks. After 8 weeks, body weight, water and food intake of mice were recorded. The level of fasting blood glucose (FBG) was measured. Serum creatinine (Scr), blood urea nitrogen (BUN), urine microalbumin (UMAlb), serum uric acid (UA) and urinary albumin excretion (UAE) were detected. An enzyme-linked immunosorbent assay was performed to test serum vascular endothelial growth factor (VEGF) and transforming growth factor-ß1 (TGF-ß1). Real-time PCR and Western blot analysis were used to test mRNA and protein expression of hypoxia inducible factor-1α (HIF-1α), VEGF and TGF-ß1 in kidney tissue. SWJH treatment significantly reduced the levels of FBG, Scr, BUN, UMAlb, UA and UAE and retarded renal fibrosis. SWJH treatment further significantly reduced serum TGF-ß1 level and downregulated the expression of HIF-1α, VEGF and TGF-ß1 at both mRNA and protein levels. Principal component analysis and partial least squares regression and hierarchical cluster analysis demonstrated that SWJH treatment significantly ameliorated renal damage in DN mice. These consequences suggested that SWJH formulations were effective in the treatment of DN through regulating the HIF-1α, VEGF and TGF-ß1 overexpression.

The demise of caterpillar fungus in the Himalayan region due to climate change and overharvesting.

Demand for traditional medicine ingredients is causing species declines globally. Due to this trade, Himalayan caterpillar fungus (Ophiocordyceps sinensis) has become one of the world's most valuable biological commodities, providing a crucial source of income for hundreds of thousands of collectors. However, the resulting harvesting boom has generated widespread concern over the sustainability of its collection. We investigate whether caterpillar fungus production is decreasing-and if so, why-across its entire range. To overcome the limitations of sparse quantitative data, we use a multiple evidence base approach that makes use of complementarities between local knowledge and ecological modeling. We find that, according to collectors across four countries, caterpillar fungus production has decreased due to habitat degradation, climate change, and especially overexploitation. Our statistical models corroborate that climate change is contributing to this decline. They indicate that caterpillar fungus is more productive under colder conditions, growing in close proximity to areas likely to have permafrost. With significant warming already underway throughout much of its range, we conclude that caterpillar fungus populations have been negatively affected by a combination of overexploitation and climate change. Our results underscore that harvesting is not the sole threat to economically valuable species, and that a collapse of the caterpillar fungus system under ongoing warming and high collection pressure would have serious implications throughout the Himalayan region.

The Effects of Dracocephalum Heterophyllum Benth Flavonoid on Hypertrophic Cardiomyocytes Induced by Angiotensin II in Rats.

BACKGROUND Dracocephalum heterophyllum Benth flavonoid (DHBF) is a Tibetan and Uighur traditional medicine used to treat various disorders such as hypertension, lung heat, cough, and bronchitis; it has good antioxidant activity. Previous studies have shown that DHBF can reduce blood pressure in renovascular hypertensive rats, improve left ventricular systolic and diastolic function, and improve myocardial contractility. Therefore, we aimed to study the effect of DHBF on cardiomyocyte hypertrophy in cultured cells. MATERIAL AND METHODS Neonatal rat cardiomyocytes were cultured, and hypertrophy was induced by angiotensin II (Ang II), with or without varying concentrations of the DHBF extract. Cell Counting Kit-8 assay was used to assess cell viability, RT-qPCR was used to determine mRNA levels, confocal laser scanning microscopy was used to measure cell surface area and intracellular Ca2+ concentrations ([Ca2+]i), and colorimetric assays were used to assess nitric oxide (NO) levels and nitric oxide synthase (NOS) activity. RESULTS Ang II treatment of cardiomyocytes reduced cell viability to ~75% that of controls. Ang II treatment also increased cell surface area; increased mRNA expression of c-jun, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and ß-myosin heavy chain (ß-MHC); increased [Ca2+]i; and reduced NOS activity and NO production. DHBF treatment could reverse these effects in a concentration-dependent manner. CONCLUSIONS These results showed that DHBF can ameliorate cardiomyocyte hypertrophy induced by Ang II, as indicated by the downregulation of cardiac hypertrophy genes (ANP, BNP, and ß-MHC) and reduction in cell surface area. The mechanism may be related to NO release and [Ca2+]I regulation.

GRGM-13 comprising 13 plant and animal products, inhibited oxidative stress induced apoptosis in retinal ganglion cells by inhibiting P2RX7/p38 MAPK signaling pathway.

OBJECTIVE: To determine the effectiveness of GRGM-13 on oxidative stress induced apoptosis of retinal ganglion cells (RGCs) and revealed its possible mechanism. MATERIALS AND METHODS: Caspase-3 activity, MDA level, and glutathione peroxidase level were detected by Caspase-3 assay kit, Lipid Peroxidation MDA Assay Kit, and Total Glutathione Peroxidase Assay Kit, respectively. Protein levels of Bax, Bcl-2, p-p38 and p38 were observed by Western Blot. Reactive oxygen species assay kit was used to determine intracellular ROS level. Apoptotic cells were measured by flow cytometry. RESULTS: GRGM-13 inhibited apoptosis of RGCs and ROS level in rat retinal tissue and RGC-5 cells, and the decrease degree strengthened with the increase of GRGM-13 concentration. In addition, ROS upregulated p-p38 expression, while GRGM-13 reversed this effect. We also found that p38 inhibitor SB202190 did not change L-glutamate (Glu) or H2O2-induced ROS level, while SB202190 inhibited apoptosis of RGC-5 cells. Finally, we observed that P2 × 7R agonist BzATP reversed the inhibition effect of GRGM-13 on RGC-5 cell apoptosis, ROS level and p-p38 expression, while si-P2 × 7R inhibited oxidative stress-induced phosphorylation of p38. CONCLUSION: GRGM-13 could inhibit oxidative stress-induced RGCs apoptosis via inhibiting P2RX7/p38 MAPK pathway, which revealed the possible mechanism of GRGM-13 on stress-induced RGCs apoptosis and provided new Chinese medicine for the treatment of glaucoma.

Systems Study on the Antirheumatic Mechanism of Tibetan Medicated-Bath Therapy Using Wuwei-Ganlu-Yaoyu-Keli.

In clinical practice at Tibetan area of China, Traditional Tibetan Medicine formula Wuwei-Ganlu-Yaoyu-Keli (WGYK) is commonly added in warm water of bath therapy to treat rheumatoid arthritis (RA). However, its mechanism of action is not well interpreted yet. In this paper, we first verify WGYK's anti-RA effect by an animal experiment. Then, based on gene expression data from microarray experiments, we apply approaches of network pharmacology to further reveal the mechanism of action for WGYK to treat RA by analyzing protein-protein interactions and pathways. This study may facilitate our understanding of anti-RA effect of WGYK from perspective of network pharmacology.